Dr. Linck's long term objective is to elucidate the function of microtubules. This proposal concerns a new class of microtubule-associated proteins, called tektins. Three tektins (A-53-kd, B-51-kd and C-47-kd) form filaments in the walls of ciliary and flagellar microtubules. Tektin filaments are associated with a highly stable segment of the microtubule wall, the protofilament (pf)-ribbon. Pf-ribbons are composed of tubulin, tektins, and other polypeptides. This proposal combines the techniques of biochemistry, structural biology, and molecular genetics to elucidate the function of tektins in microtubules from sea urchin sperm and Chlamydomonas reinhardtii. Aim 1 is to produce a molecular model of pf-ribbons. Tektin polymers will be reconstituted from purified subunits and analyzed biochemically and by EM to determine whether tektins A, B, and C form homopolymers or heteropolymers. Chemical crosslinking procedures will be used to map associations with neighboring polypeptides. EM, cryo-EM and STEM will be used to study the molecular arrangements of the proteins forming pf- ribbons. Aim 2 is to identify and characterize tektin genes and proteins in Chlamydomonas. Cloned genomic and/or cDNA sequences will be determined and analyzed for their gene structure, predicted protein structure, and potential tubulin binding domains.Tektin proteins will be purified and characterized in order to confirm the DNA sequences and to construct oligonucleotides for PCR screening. Gene specific probes will be used to determine the number of tektin genes and their location on the genetic map. If tektin genes map to known loci for flagellar defects, the mutant proteins will be characterized to identify the defects. Aim 3 is to investigate the function of tektins in Chlamydomonas using molecular/genetic and cell biological approaches. The in vivo function(s) of tektins will be studied by regulating the expression of native or mutant tektins in wild-type and mutant backgrounds. The assembly of epitope-tagged tektins will be followed in regenerating flagella. Finally, anti-tektin antibodies will be used to precipitate possible oligomeric tektin-tubulin intermediates and/or phosphorylated tektins.